Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. The disease has also been considered by some as a segmental progeriod entity, as FA patients develop a range of tissue-specific premature onset and accelerated aging phenotypes, including bilateral cataracts, skin atrophy, reduced muscle mass, premature ovarian failure, higher frequency of osteoporosis and osteopenia, and diabetes. Fifteen genes have now been described that are mutated to cause FA, three discovered by our group. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in that pathway is modification of two FA proteins, FANCD2 and FANCI. The modification, monoubiquitylation, results in redistribution of FANCD2-FANCI to specific spots in the nucleus where BRCA proteins also localize. When we initiated this project in 2001, five FA proteins (FANCA, -C, -E, -F, and -G) were found to interact with each other to form a multiprotein nuclear complex, the FA core complex. This complex functions upstream in the pathway and is required for FANCD2-FANCI monoubiquitylation. We purified the FA core complex and found that it contains at least seven new components in addition to the five known FA proteins. We have characterized these new components and shown that they are important for the FA-associated DNA damage response pathway, as summarized below. One new component of the FA core complex, termed PHF9, possesses ubiquitin ligase activity in vitro and is essential for FANCD2-FANCI monoubiquitylation in vivo. PHF9 is defective in a group of Fanconi anemia patients, and therefore represents a novel Fanconi anemia gene (FANCL). Our data suggest that PHF9 plays a crucial role in the Fanconi anemia pathway as the catalytic subunit for monoubiquitylation and FANCD2-FANCI. The discovery of PHF9/FANCL might provide a potential target for new therapeutic modalities. We then showed that the 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). FANCB is X-linked and present in only one active copy. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability, because it will take only one mutation to inactivate FANCB in males, compared to two mutations required to inactivate other autosomal FA genes. We demonstrated that the 250 Kda subunit of the FA core complex is mutated in FA patients of a new complementation group, FA-M. The gene was named FANCM. FANCM has a conserved helicase domain and a DNA remodeling activity. FANCM has at least three important roles in the FA DNA damage response pathway. First, it plays a structural role, allowing assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM translocates and remodels various DNA structures, which are important for subsequent DNA repair. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. We have identified the 100 Kda subunit of the FA core complex, FAAP100, and shown that this protein is required for stability and a key function of the complexmonoubiquitination of FANCD2-FANCI. We have identified the 24 Kda subunit of the FA core complex, termed FAAP24, which forms a heterodimer with FANCM. FAAP24 can recognize structured DNA that mimics intermediates generated during DNA replication. Moreover, it can target FANCM to such structures. Cells depleted of FAAP24 show phenotypes that are characteristics of FA cells. Our results demonstrate that FAAP24 is an integral component of the FA core complex, We also collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. In further mechanistic studies, we demonstrated that FANCM possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. We found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA crosslinking drug, mitomycin C (MMC), but not for the monoubiquitination of FANCD2-FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP- dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair. We identified two new components in the FA core complex, MHF1 and MHF2. These two proteins form a histone-fold heterodimer that associates with FANCM to form a DNA-remodeling complex conserved from yeast to human. MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human. We showed that another FA protein, FANCJ, becomes hyperphosphorylated in response to DNA damage. We are investigating if this phosphorylation regulates activity of FANCJ in DNA repair. We collaborated with Dr. Lei Li's lab to develop a chromatin-IP-based strategy termed eChIP and elucidated how various FA proteins are recruited to the interstrand DNA crosslinks that block replication. We found that BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. We have recently identified FAAP20 as a new component of the FA core complex. FAAP20 contains an ubiquitin binding motif and is required for FA core complex to localize to DNA damage sites. Depletion or inactivation of FAAP20 reduces monoubiquitylation of FANCD2-FANCI, indicating that FAAP20 is an important player of the FA pathway. The fact that FANCM and MHF1-MHF2 constitute a conserved DNA remodeling machine prompted us to perform crystal structure studies of this complex. So far, we have also solved the crystal structure of MHF1-MHF2 complex in conjunction with the region of FANCM that binds MHF. The structure reveals how these three proteins interact with each other to form a complex and recognizes DNA. We are continuing to identify and characterize additional components of the FA complex. The eventual goal is to analyze the full pathway of DNA crosslink repair, assess its role during normal aging, and in DNA repair-deficient diseases.